Lens membrane fraction associated intermediate filaments of different aged rats

Abstract

Purpose. To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. Methods. Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. Results. Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. Conclusions. The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane–cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.